A crystal form of a pyrroloheterocyclic derivative and a preparation method thereof

Abstract

Provided are a crystalline form of a pyrroloheterocyclic derivative and a method for preparing the same. Specifically, the invention relates to a crystalline form of a pharmaceutically acceptable salt of a compound represented by formula (I) and a method for preparing the same. The crystalline form of the pharmaceutically acceptable salt of the compound represented by formula (I) provided has good stability and can be better used in clinical treatment.

Description

[0001] This application claims the priority of Chinese Patent Application No. 2020110494022, filed on September 29, 2020. This application cites the entire text of the above Chinese Patent Application. Technical Field

[0002] The present invention relates to a novel crystal form of a pyrroloheterocyclic derivative and a preparation method thereof, and belongs to the pharmaceutical field. Background Art

[0003] The proliferation, differentiation, metabolism, and apoptosis of normal cells are strictly regulated by the cell signal transduction pathway in the body. Mitogen-activated protein kinase (MAPK) plays an extremely important role in the signal transduction pathway, and extracellular signal regulated kinase (ERK) is a member of the MAPK family. Through the RAS-RAF-MEK-ERK step, the exogenous stimulation signal is transmitted to ERK, and the activated ERK transfers into the cell nucleus to regulate the activity of transcription factors, thereby regulating the cell's proliferation, differentiation, apoptosis and other biological functions, or participating in the regulation of cell morphology and the redistribution of the cytoskeleton by phosphorylating the cytoskeletal components in the cytoplasm.

[0004] Mutations in the RAS and RAF genes cause the continuous activation of the MAPK-ERK signaling pathway, which promotes malignant transformation and abnormal proliferation of cells, and ultimately produces tumors (Roberts PJ et al., Oncogene, 2007, 26 (22), 3291-3310). The combination of MEK inhibitors and B-RAF inhibitors can further enhance the effect of B-RAF inhibitors in inhibiting tumor growth, and can significantly improve the disease-free progression period and overall survival rate of melanoma patients carrying BRAFV600E and V600K mutations (Frederick DT et al., Clinical Cancer Research, 2013. 19 (5), 1225-1231). Although the combination of B-RAF / MEK inhibitors can inhibit tumors, their efficacy is short-lived. Within 2-18 months, most patients will develop drug resistance and the tumor will further deteriorate. The mechanism of resistance to B-RAF / MEK inhibitors is very complex, and most of them are directly related to the reactivation of the ERK signaling pathway (Smalley I et al., Cancer Discovery, 2018, 8(2), 140-142). Therefore, the development of new ERK inhibitors is not only effective for patients with mutations in the MAPK signaling pathway, but also for patients with resistance to B-RAF / MEK inhibitors.

[0005] B-RAF / MEK inhibitors not only inhibit tumor growth, but also regulate the immune microenvironment of tumors. B-RAF / MEK inhibitors can enhance the expression of tumor-specific antigens, improve the recognition and killing of tumors by antigen-specific T cells, and promote the migration and infiltration of immune cells. In animal models, after treatment with B-RAF / MEK inhibitors, PD-L1 expression in tumor tissues is enhanced. When used in combination with antibodies to checkpoint molecules (e.g., PD-1 antibodies, CTLA4 antibodies), it shows an effect of inhibiting tumor growth that is superior to that of B-RAF / MEK inhibitors alone (Boni A et al., Cancer Research, 2010, 70(13), 5213-5219). Studies have shown that ERK inhibitors are similar to B-RAF / MEK inhibitors. When used in combination with checkpoint antibodies, they can regulate the tumor microenvironment, improve the function of cytotoxic T cells, and achieve the effect of inhibiting tumor growth.

[0006] Currently, several compounds have been developed. Among them, BVD-523 of BioMed Valley Discoveries is in Phase II clinical trials, MK-8353 of Merck, and Astex-029 of Astex are in Phase I clinical trials. Related patents include WO1999061440A1, WO2001056557A2, WO2001056993A2, WO2001057022A2, WO2002022601A1, WO2012118850A1, WO2013018733A1, WO2014179154A2, WO2015103133A1, WO2016192063A1, WO2017180817A1, and WO2018049127A1. Summary of the invention

[0007] The present disclosure provides a pharmaceutically acceptable salt of a compound represented by formula (I), wherein the pharmaceutically acceptable salt is selected from fumarate, oxalate, succinate, maleate, hydrochloride or hydrobromide.

[0008]

[0009] The present disclosure further provides a method for preparing a pharmaceutically acceptable salt of a compound of formula (I), comprising the step of reacting the compound of formula (I) with a fumarate, oxalate, succinate, maleate, hydrochloride or hydrobromide.

[0010] In an optional embodiment, the present disclosure provides a fumarate salt of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to fumaric acid is 1:1 or 2:1.

[0011] The present disclosure further provides a method for preparing a fumarate salt of the compound represented by formula (I), comprising the step of reacting the compound represented by formula (I) with fumaric acid.

[0012] Mu Gongkai further provides a crystalline form I of a fumarate of a compound represented by formula (I), wherein the fumarate of a compound represented by formula (I) has a molar ratio of the compound represented by formula (I) to fumaric acid of 1:1, and an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, having characteristic peaks at 10.671, 22.735, 28.681, and 29.302.

[0013] The present disclosure further provides a crystalline form I of a fumarate of a compound represented by formula (I), wherein the fumarate of the compound represented by formula (I) has a molar ratio of the compound represented by formula (I) to fumaric acid of 1:1, and an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, having characteristic peaks at 10.671, 16.228, 19.832, 22.735, 23.526, 28.681, and 29.302.

[0014] Another aspect of the present disclosure provides a method for preparing a crystalline form I of a fumarate salt of a compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to fumaric acid is 1:1, and the method comprises the following steps:

[0015] 1) dissolving the compound represented by formula (I) and fumaric acid in a solvent selected from ethyl acetate,

[0016] 2) crystallization, wherein the molar ratio of the compound represented by formula (I) to fumaric acid is selected from 1:1 to 1.5.

[0017] The present disclosure further provides a crystal form II of a fumarate of a compound represented by formula (I), wherein the fumarate of the compound represented by formula (I) has a molar ratio of the compound represented by formula (I) to fumaric acid of 1:1, and an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, having characteristic peaks at 7.783, 9.151, 12.378, 14.808, 18.574, 25.755, and 26.802.

[0018] In an optional embodiment, the present disclosure provides a crystal form II of a fumarate salt of a compound represented by formula (I), wherein the fumarate salt of a compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to fumaric acid is 1:1, and the X-ray powder diffraction pattern represented by a diffraction angle of 2θ has characteristic peaks at 7.783, 9.151, 10.292, 12.378, 14.808, 18.574, 19.632, 20.677, 22.305, 23.394, 25.755, 26.802, and 27.684.

[0019] In an optional embodiment, the crystal form II of the fumarate of the compound represented by formula (I) provided by Mu Dong, the fumarate of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to fumaric acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 7.783, 9.151, 10.292, 12.378, 14.808, 16.386, 18.574, 19.632, 20.677, 22.305, 23.394, 24.053, 24.495, 25.755, 26.802, 27.684, 28.877, 29.681, and 31.851.

[0020] Another aspect of the present disclosure provides a method for preparing a crystal form II of a fumarate salt of a compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to fumaric acid is 1:1, and the method comprises the following steps:

[0021] 1) dissolving the compound represented by formula (I) and fumaric acid in ethanol or acetonitrile solution,

[0022] 2) crystallization, wherein the molar ratio of the compound represented by formula (I) to fumaric acid is selected from 1:1 to 1.5.

[0023] In an optional embodiment, the present disclosure provides a III crystalline form of a fumarate salt of a compound represented by formula (I), wherein the fumarate salt of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to fumaric acid is 2:1, and the X-ray powder diffraction pattern represented by a diffraction angle of 2θ has characteristic peaks at 4.877, 10.601, 13.827, 14.867, 16.272, 18.874, 23.921, and 26.367.

[0024] In an optional embodiment, the present disclosure provides a III crystalline form of a fumarate salt of a compound represented by formula (I), wherein the fumarate salt of a compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to fumaric acid is 2:1, and an X-ray powder diffraction pattern represented by a diffraction angle of 2θ has characteristic peaks at 4.877, 9.664, 10.601, 11.745, 13.827, 14.867, 16.272, 17.885, 18.874, 20.695, 21.320, 22.204, 23.921, and 26.367.

[0025] In an optional embodiment, the present disclosure provides a crystal form IV of a fumarate salt of a compound represented by formula (I), wherein the fumarate salt of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to fumaric acid is 2:1, and the X-ray powder diffraction pattern represented by a diffraction angle of 2θ has characteristic peaks at 4.854, 10.613, 13.692, 23.523, 24.252, and 25.925.

[0026] In an optional embodiment, the present disclosure provides a crystal form IV of a fumarate salt of a compound represented by formula (I), wherein the fumarate salt of a compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to fumaric acid is 2:1, and an X-ray powder diffraction pattern represented by a diffraction angle of 2θ has characteristic peaks at 4.854, 9.785, 10.613, 13.692, 14.794, 18.424, 19.107, 20.409, 23.523, 24.252, and 25.925.

[0027] In an optional embodiment, the present disclosure provides a crystal form IV of a fumarate salt of a compound of formula (I), wherein the molar ratio of the compound of formula (I) to fumaric acid is 2:1, and an X-ray powder diffraction pattern represented by a diffraction angle of 2θ has characteristic peaks at 4.854, 8.650, 9.785, 10.613, 11.658, 12.449, 13.692, 14.794, 15.393, 16.257, 17.864, 18.424, 19.107, 20.409, 21.322, 22.510, 23.523, 24.252, 25.925, and 28.382.

[0028] A method for preparing a crystal form IV of a fumarate of a compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to fumaric acid is 2:1, and the crystal form III of the fumarate of the compound represented by formula (I) is placed in an environment with a humidity greater than 50%.

[0029] In an optional embodiment, the present disclosure provides an oxalate salt of the compound represented by formula (I).

[0030] The present disclosure further provides a crystalline form a of the oxalate of the compound represented by formula (I), and an X-ray powder diffraction pattern represented by a diffraction angle 2θ has characteristic peaks at 14.235, 15.983, 17.882, and 26.923.

[0031] In an optional embodiment, the present disclosure provides a maleate salt of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to maleic acid is 1:1.

[0032] The present disclosure further provides a crystalline form a of a maleate of a compound represented by formula (I), wherein the maleate of a compound represented by formula (I) has a molar ratio of the compound represented by formula (I) to maleic acid of 1:1, and an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, having characteristic peaks at 4.510, 18.170, 23.973, 24.864, 26.306, and 28.209.

[0033] In an optional embodiment, the present disclosure provides a crystalline form a of the maleate salt of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to maleic acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 4.510, 18.170, 19.959, 21.842, 23.973, 24.864, 26.306, 28.209, and 31.995.

[0034] On the other hand, the present disclosure provides a method for preparing a crystalline form of a maleate salt of a compound represented by formula (I), wherein the maleate salt of the compound represented by formula (I) has a molar ratio of the compound represented by formula (I) to maleic acid of 1:1, and the method comprises the following steps: 1) dissolving the compound represented by formula (I) and maleic acid in an isopropanol solution, and 2) crystallizing, wherein the molar ratio of the compound represented by formula (I) to maleic acid is selected from 1:1 to 1.5.

[0035] The present disclosure further provides a b-form of a maleate salt of a compound represented by formula (I), wherein the maleate salt of the compound represented by formula (I) has a molar ratio of the compound represented by formula (I) to maleic acid of 1:1, and an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, having characteristic peaks at 4.744, 10.024, 11.931, 15.969, 19.171, 25.291, and 28.006.

[0036] In an optional embodiment, the present disclosure provides a b-form of the maleate salt of the compound represented by formula (I), wherein the maleate salt of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to maleic acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 4.744, 10.024, 11.931, 14.420, 15.185, 15.969, 17.249, 19.171, 20.405, 24.110, 25.291, 28.006, 30.216, and 32.380.

[0037] On the other hand, the present disclosure provides a method for preparing the b-crystalline form of the maleate salt of the compound represented by formula (I), wherein the maleate salt of the compound represented by formula (I) has a molar ratio of the compound represented by formula (I) to maleic acid of 1:1, and the method comprises the following steps: 1) dissolving the compound represented by formula (I) and maleic acid in an ethyl acetate solution, and 2) crystallizing, wherein the molar ratio of the compound represented by formula (I) to maleic acid is selected from 1:1 to 1.5.

[0038] The present disclosure further provides a crystal form C of a maleate of a compound represented by formula (I), wherein the maleate of the compound represented by formula (I) has a molar ratio of the compound represented by formula (I) to maleic acid of 1:1, and an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, having characteristic peaks at 4.610, 9.840, 10.708, 11.414, 12.329, 18.485, 18.883, 19.328, and 26.160.

[0039] In an optional embodiment, the present disclosure provides a maleate salt of a compound of formula (I) in form C, wherein the maleate salt of the compound of formula (I), wherein the molar ratio of the compound of formula (I) to maleic acid is 1:1, has an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, having characteristic peaks at 4.610, 9.840, 10.708, 11.414, 12.329, 14.346, 15.262, 16.655, 17.298, 18.485, 18.883, 19.328, 23.928, 26.160, and 28.389.

[0040] In an optional embodiment, the present disclosure provides a maleate salt of a compound of formula (I) in form C, wherein the maleate salt of the compound of formula (I), wherein the molar ratio of the compound of formula (I) to maleic acid is 1:1, has an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, and has characteristic peaks at 4.610, 9.840, 10.708, 11.414, 12.329, 12.395, 14.346, 15.262, 15.962, 16.655, 17.298, 18.485, 18.883, 19.328, 22.250, 23.928, 26.160, 28.389, and 31.162.

[0041] On the other hand, the present disclosure provides a method for preparing the C crystal form of the maleate salt of the compound represented by formula (I), wherein the maleate salt of the compound represented by formula (I) has a molar ratio of the compound represented by formula (I) to maleic acid of 1:1, and the method comprises the following steps: 1) dissolving the compound represented by formula (I) and maleic acid in an acetonitrile solution, 2) crystallizing, and the molar ratio of the compound represented by formula (I) to maleic acid is selected from 10 to 2:1.

[0042] The present disclosure further provides a d-form of a maleate salt of a compound represented by formula (I), wherein the maleate salt of the compound represented by formula (I) has a molar ratio of the compound represented by formula (I) to maleic acid of 1:1, and an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, having characteristic peaks at 6.679, 9.346, 13.476, 14.076, 15.388, and 19.183.

[0043] In an optional embodiment, the present disclosure provides a d-form of the maleate salt of the compound represented by formula (I), wherein the maleate salt of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to maleic acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 6.679, 8.405, 9.346, 10.170, 13.476, 14.076, 15.388, 19.183, 21.228, and 26.247.

[0044] In an optional embodiment, the present disclosure provides a d-form of the maleate salt of the compound of formula (I), wherein the molar ratio of the compound of formula (I) to maleic acid is 1:1, and the X-ray powder diffraction pattern expressed as a diffraction angle 2θ has characteristic peaks at 6.679, 8.405, 9.346, 10.170, 10.876, 13.476, 14.076, 15.388, 16.993, 19.183, 20.286, 21.228, 22.286, 23.894, 24.639, 26.247, 27.070, and 28.403.

[0045] The present disclosure further provides a crystal form e of a maleate of a compound represented by formula (I), wherein the maleate of the compound represented by formula (I) has a molar ratio of the compound represented by formula (I) to maleic acid of 1:1, and an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, having characteristic peaks at 6.193, 10.009, 13.646, 17.582, 17.996, 18.758, 22.733, and 24.766.

[0046] In an optional embodiment, the present disclosure provides the e-crystalline form of the maleate salt of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to maleic acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 6.193, 10.009, 13.646, 16.375, 17.582, 17.996, 18.758, 19.306, 20.181, 22.733, and 24.766.

[0047] In an optional embodiment, the present disclosure provides the e-crystalline form of the maleate salt of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to maleic acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 6.193, 10.009, 11.591, 13.646, 16.375, 17.582, 17.996, 18.758, 19.306, 20.181, 22.733, 24.766, 25.332, 26.601, 27.303, 28.859, and 31.447.

[0048] In an optional embodiment, the present disclosure provides a succinate salt of the compound represented by formula (I).

[0049] The present disclosure further provides an α-crystalline form of a succinate salt of a compound represented by formula (I), and an X-ray powder diffraction pattern represented by a diffraction angle 2θ has characteristic peaks at 5.640, 10.720, 13.384, 15.208, 16.244, 19.992, 21.669, 23.839, and 24.826.

[0050] In an optional embodiment, the α-crystalline form of the succinate salt of the compound represented by formula (I) provided by the present disclosure has an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, with characteristic peaks at 5.640, 10.720, 13.384, 15.208, 16.244, 17.280, 18.020, 18.907, 19.992, 21.669, 23.839, and 24.826.

[0051] The present disclosure further provides a β-crystalline form of a succinate salt of a compound represented by formula (I), and an X-ray powder diffraction pattern represented by a diffraction angle 2θ has characteristic peaks at 5.160, 17.951, 20.618, 25.557, and 26.545.

[0052] In an optional embodiment, the present disclosure provides a hydrobromide salt of a compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to hydrobromic acid is 1:1.

[0053] Furthermore, the present disclosure provides an α-crystalline form of the hydrobromide of the compound represented by the above formula (I), wherein the hydrobromide of the compound represented by the formula (I), wherein the molar ratio of the compound represented by the formula (I) to hydrobromic acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 6.656, 9.552, 14.110, 19.095, 20.092, 25.220, and 27.119.

[0054] In an optional embodiment, the present disclosure provides an α-crystalline form of the hydrobromide of the compound represented by formula (I), wherein the hydrobromide of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to hydrobromic acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 6.656, 9.552, 13.778, 14.110, 19.095, 19.522, 20.092, 21.422, 25.220, 25.837, and 27.119.

[0055] In an optional embodiment, the present disclosure provides an α-crystalline form of the hydrobromide of the compound represented by formula (I), wherein the hydrobromide of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to hydrobromic acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 6.656, 9.552, 13.778, 14.110, 19.095, 19.522, 20.092, 21.422, 22.276, 22.988, 23.653, 25.220, 25.837, 26.217, 27.119, 31.107, 31.867, 32.769, and 34.810.

[0056] In an optional embodiment, the present disclosure provides a hydrobromide salt of a compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to hydrobromic acid is 1:2.

[0057] The present disclosure provides a method for preparing a hydrobromide salt of a compound represented by formula (I), comprising the step of reacting the compound represented by formula (I) with a hydrobromide salt.

[0058] Furthermore, the present disclosure provides a β-crystalline form of the hydrobromide of the compound represented by the above formula (I), wherein the hydrobromide of the compound represented by the formula (I), wherein the molar ratio of the compound represented by the formula (I) to hydrobromic acid is 1:2, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 7.363, 12.914, 15.392, 16.865, 17.217, 19.704, 21.999, 22.515, 24.769, 27.056, and 28.236.

[0059] In an optional embodiment, the present disclosure provides a β-crystalline hydrobromide of a compound of formula (I), wherein the molar ratio of the compound of formula (I) to hydrobromic acid is 1:2, and an X-ray powder diffraction pattern expressed as a diffraction angle 2θ has characteristic peaks at 7.363, 12.914, 14.351, 15.392, 16.865, 17.217, 19.704, 21.999, 22.515, 24.769, 27.056, 28.236, and 29.234.

[0060] In an optional embodiment, the present disclosure provides a β-crystalline hydrobromide of a compound of formula (I), wherein the molar ratio of the compound of formula (I) to hydrobromic acid is 1:2, and an X-ray powder diffraction pattern expressed as a diffraction angle 2θ has characteristic peaks at 7.363, 12.914, 14.351, 15.392, 16.865, 17.217, 19.452, 19.704, 21.999, 22.515, 23.275, 24.213, 24.769, 27.056, 28.236, 29.234, 29.764, 31.251, and 32.887.

[0061] In an optional embodiment, the present disclosure provides a γ-crystalline form of the hydrobromide of the compound represented by the above formula (I), wherein the hydrobromide of the compound represented by the formula (I), wherein the molar ratio of the compound represented by the formula (I) to hydrobromic acid is 1:2, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 5.463, 16.567, 21.227, 25.784, 26.643, 28.321, and 33.788.

[0062] In an optional embodiment, the present disclosure provides a γ-crystalline form of the hydrobromide of the compound represented by formula (I), wherein the hydrobromide of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to hydrobromic acid is 1:2, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 5.463, 16.567, 19.733, 21.227, 22.332, 25.014, 25.784, 26.643, 28.321, 30.298, 32.170, 33.788, 36.211, and 37.939.

[0063] In an optional embodiment, the present disclosure provides a hydrochloride salt of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to hydrochloric acid is 1:1.

[0064] Furthermore, the present disclosure provides a crystalline form I of the hydrochloride of the compound represented by the above formula (I), wherein the hydrochloride of the compound represented by the formula (I), wherein the molar ratio of the compound represented by the formula (I) to hydrochloric acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 5.187, 9.407, 9.848, 10.388, 17.354, 18.041, and 26.234.

[0065] In an optional embodiment, the present disclosure provides Form I of the hydrochloride of the compound represented by formula (I), wherein the hydrochloride of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to hydrochloric acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 5.187, 8.131, 9.407, 9.848, 10.388, 17.354, 18.041, 18.924, 23.830, 25.449, 26.234, 26.724, 27.362, 27.902, 29.275, and 31.581.

[0066] Furthermore, the present disclosure provides a crystal form II of the hydrochloride of the compound represented by the above formula (I), wherein the hydrochloride of the compound represented by the formula (I), wherein the molar ratio of the compound represented by the formula (I) to hydrochloric acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 13.728, 17.948, 22.678, 25.525, 27.356, and 29.543.

[0067] In an optional embodiment, the present disclosure provides a crystalline form II of the hydrochloride of the compound represented by formula (I), wherein the hydrochloride of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to hydrochloric acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 13.728, 17.948, 20.593, 22.678, 25.525, 27.356, and 29.543.

[0068] Furthermore, the present disclosure provides a III crystal form of the hydrochloride of the compound represented by the above formula (I), wherein the hydrochloride of the compound represented by the formula (I), wherein the molar ratio of the compound represented by the formula (I) to hydrochloric acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 5.854, 8.983, 11.902, 13.181, 18.159, 20.071, 21.371, 24.180, 25.066, 26.469, 27.086, 27.729, and 28.615.

[0069] In an optional embodiment, the present disclosure provides a III crystal form of the hydrochloride of the compound represented by formula (I), wherein the hydrochloride of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to hydrochloric acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 5.854, 8.983, 11.902, 13.181, 16.876, 18.159, 20.071, 21.371, 22.799, 24.180, 25.066, 26.469, 27.086, 27.729, 28.615, 29.827, 30.904, 31.935, 33.571, 34.905, and 37.265.

[0070] Furthermore, the present disclosure provides a crystalline form IV of the hydrochloride of the compound represented by the above formula (I), wherein the hydrochloride of the compound represented by the formula (I), wherein the molar ratio of the compound represented by the formula (I) to hydrochloric acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 5.693, 12.474, 14.474, 17.840, 22.231, 23.938, 26.866, and 29.207.

[0071] In an optional embodiment, the IV crystal form of the hydrochloride of the compound represented by formula (I) provided by the present disclosure, the hydrochloride of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to hydrochloric acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 5.693, 12.474, 14.474, 17.352, 17.840, 19.596, 22.231, 23.109, 23.938, 24.573, 26.866, 27.841, 29.207, 33.452, and 34.915.

[0072] Furthermore, the present disclosure provides a V crystal form of the hydrochloride of the compound represented by the above formula (I), wherein the hydrochloride of the compound represented by the formula (I), wherein the molar ratio of the compound represented by the formula (I) to hydrochloric acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 7.476, 14.346, 15.181, 17.185, 21.606, 22.770, 24.279, 24.952, and 28.336.

[0073] In an optional embodiment, the present disclosure provides a V crystal form of the hydrochloride of the compound represented by formula (I), wherein the hydrochloride of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to hydrochloric acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 7.476, 9.536, 13.447, 14.346, 15.181, 17.185, 18.422, 21.606, 22.770, 24.279, 24.952, 28.336, and 29.773.

[0074] In an optional embodiment, the V crystal form of the hydrochloride of the compound represented by formula (I) provided by the present disclosure, the hydrochloride of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to hydrochloric acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 7.476, 9.536, 12.349, 13.447, 14.346, 15.181, 15.639, 16.694, 17.185, 18.422, 18.859, 21.606, 22.770, 23.425, 24.279, 24.952, 26.990, 28.336, 28.772, 29.773, and 30.846.

[0075] In an optional embodiment, the crystalline form of the pharmaceutically acceptable salt of the compound represented by the aforementioned formula (I), wherein the error range of the 2θ angle is ±0.3.

[0076] In an optional embodiment, the crystalline form of the pharmaceutically acceptable salt of the compound represented by the aforementioned formula (I), wherein the error range of the 2θ angle is ±0.2.

[0077] In certain embodiments, the method for preparing the crystalline form described in the present disclosure further comprises filtering, washing or drying steps.

[0078] The present disclosure also provides a pharmaceutical composition prepared from a pharmaceutically acceptable salt of the compound represented by the aforementioned formula (I) or a crystalline form of a pharmaceutically acceptable salt.

[0079] The present disclosure also provides a pharmaceutical composition comprising the following components: i) a pharmaceutically acceptable salt of the compound represented by the aforementioned formula (I), or a crystalline form of a pharmaceutically acceptable salt, and ii) a pharmaceutically acceptable carrier, diluent or excipient.

[0080] The present disclosure also provides a method for preparing a pharmaceutical composition, comprising the step of mixing the aforementioned component i) and component ii).

[0081] The present disclosure also provides a pharmaceutically acceptable salt of the compound represented by the aforementioned formula (I), or a crystalline form of a pharmaceutically acceptable salt of the compound represented by the aforementioned formula (I), or the aforementioned composition, or use of the composition prepared by the aforementioned method in preparing a medicament for treating or preventing cancer, inflammation, or other proliferative diseases, preferably cancer; the cancer is selected from melanoma, liver cancer, kidney cancer, lung cancer, nasopharyngeal cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, head and neck squamous cell carcinoma, cervical cancer, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma, astrocytoma and glioma.

[0082] The "2θ or 2θ angle" described in the present disclosure refers to the diffraction angle, θ is the Bragg angle, and the unit is ° or degree; the error range of each characteristic peak 2θ is ±0.20, which can be -0.20, -0.19, -0.18, -0.17, -0.16, -0.15, -0.14, -0.13, -0.12, -0.11, -0.10, -0.09, -0.08, -0.07, -0.06, -0.05, -0.04, -0.03, -0.02, -0.01, 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20.

[0083] The "crystallization" described in the present disclosure includes but is not limited to stirring crystallization, slurry crystallization and volatilization crystallization.

[0084] The drying temperature in the present disclosure is generally 25° C.-100° C., preferably 40° C.-70° C., and the drying can be performed under normal pressure or reduced pressure.

[0085] In the present application, the step of reacting the compound represented by formula (I) with fumarate, oxalate, succinate, maleate, hydrochloride or hydrobromide can be optionally carried out in an appropriate solvent, such as an ester solvent, specifically ethyl acetate; such as an alcohol solvent, specifically methanol, ethanol or isopropanol; such as a nitrile solvent, specifically acetonitrile and propionitrile; such as a ketone solvent, specifically acetone; such as an ether solvent, specifically tetrahydrofuran, dioxane, diethyl ether, isopropyl ether; such as an alkane solvent, specifically n-hexane, n-heptane, or water, or a mixed solvent of the above solvents. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 . The amorphous XRPD spectrum of the compound represented by formula (I);

[0087] Figure 2. XRPD spectrum of Form I of the fumarate salt of the compound represented by formula (I);

[0088] Figure 3 . XRPD spectrum of Form II of the fumarate salt of the compound represented by formula (I);

[0089] Figure 4 DSC spectrum of the crystal form II of the fumarate salt of the compound represented by formula (I)

[0090] Figure 5 . TGA spectrum of the II crystal form of the fumarate salt of the compound represented by formula (I)

[0091] Figure 6 .DVS spectrum of the II crystal form of the fumarate salt of the compound represented by formula (I)

[0092] Figure 7 XRPD spectra of Form II of the fumarate salt of the compound represented by formula (I) before and after DVS;

[0093] Figure 8 . XRPD spectrum of Form III of the fumarate salt of the compound represented by formula (I);

[0094] Fig. 9 . XRPD spectrum of Form IV of the fumarate salt of the compound represented by formula (I);

[0095] Fig.10 . XRPD spectrum of the a-crystalline form of the oxalate salt of the compound represented by formula (I);

[0096] Fig.11 . XRPD spectrum of the maleate salt of the compound represented by formula (I) in Form a;

[0097] Fig.12 . XRPD spectrum of the b-form of the maleate salt of the compound represented by formula (I);

[0098] Fig.13 . XRPD spectrum of Form C of the maleate salt of the compound represented by Formula (I);

[0099] Fig.14 . An amorphous XRPD spectrum of the maleate salt of the compound represented by formula (I);

[0100] Fig.15 . XRPD spectrum of the d-form of the maleate salt of the compound represented by formula (I);

[0101] Fig.16 . XRPD spectrum of the crystal form e of the maleate salt of the compound represented by formula (I);

[0102] Fig.17. XRPD spectrum of the α-crystalline form of the succinate salt of the compound represented by formula (I);

[0103] Fig.18 . XRPD spectrum of the β-crystalline form of the succinate salt of the compound represented by formula (I);

[0104] Fig.19 XRPD spectrum of the α-crystalline form of the hydrobromide salt of the compound represented by formula (I);

[0105] Fig. 20 . XRPD spectrum of the β-crystalline form of the hydrobromide salt of the compound represented by formula (I);

[0106] Fig.21 . XRPD spectrum of the γ-crystalline form of the hydrobromide salt of the compound represented by formula (I);

[0107] Fig. 22 . XRPD spectrum of Form I of the hydrochloride salt of the compound represented by formula (I);

[0108] Fig.23 . XRPD spectrum of Form II of the hydrochloride salt of the compound represented by formula (I);

[0109] Fig.24 XRPD spectrum of Form III of the hydrochloride salt of the compound represented by formula (I);

[0110] Fig.25 . XRPD spectrum of Form IV of the hydrochloride salt of the compound represented by formula (I);

[0111] Fig.26 . XRPD spectrum of Form V of the hydrochloride salt of the compound represented by formula (I). DETAILED DESCRIPTION

[0112] The present invention will be explained in more detail below in conjunction with embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention, and are not intended to limit the essence and scope of the present invention.

[0113] Test conditions of the instruments used in the experiment:

[0114] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10 -6 The NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer or a Bruker AVANCE NEO 500M NMR spectrometer, and the solvent used was deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), and the internal standard was tetramethylsilane (TMS).

[0115] MS was measured using an Agilent 1200 / 1290 DAD-6110 / 6120 Quadrupole MS liquid spectrometer (manufacturer: Agilent, MS model: 6110 / 6120 Quadrupole MS).

[0116] waters ACQuity UPLC-QD / SQD (Manufacturer: waters, MS model: waters ACQuity QdaDetector / waters SQ Detector)

[0117] THERMO Ultimate 3000-Q Exactive (Manufacturer: THERMO, MS model: THERMOQExactive)

[0118] High performance liquid chromatography (HPLC) analysis was performed using Agilent HPLC 1200DAD, Agilent HPLC 1200VWD and Thermo U3000 DAD high pressure liquid chromatographs.

[0119] Chiral HPLC analysis was performed using an Agilent 1260 DAD high performance liquid chromatograph.

[0120] High performance liquid chromatography was performed using Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson GX-281 preparative chromatographs.

[0121] Chiral preparations were performed using a Shimadzu LC-20AP preparative chromatograph.

[0122] Ion chromatography was performed using Thermo Scientific Dionex Intergrion, column model: Dionex IonPacTM AS11-HC (4 μm, 4×250 cm). XRPD is X-ray powder diffraction detection: the measurement was performed using a BRUKERD8 Discovery X-ray diffractometer, specific collection information: Cu anode (40 kV, 40 mA), Cu-Kα ray Scanning mode: θ / 2θ, scanning range (2q range): 5~50°.

[0123] DSC is differential scanning calorimetry: the measurement was performed using a METTLER TOLEDO DSC 3+ differential scanning calorimeter, with a heating rate of 10°C / min, the specific temperature range refers to the corresponding spectrum (mostly 25-300 or 25-350°C), and a nitrogen purge rate of 50 mL / min.

[0124] TGA is thermogravimetric analysis: the test was performed using a METTLER TOLEDO TGA 2 thermogravimetric analyzer, with a heating rate of 10°C / min, the specific temperature range referred to the corresponding spectrum (mostly 25-300°C), and a nitrogen purge rate of 50 mL / min.

[0125] DVS stands for dynamic moisture adsorption: SMS DVS Advantage is used for detection. At 25°C, the humidity changes from 50% to 95% to 0% to 95% to 50%, with a step of 10% (the last step is 5%) (the specific range of humidity is subject to the corresponding spectrum, and most of the methods listed here are used). The judgment standard is that dm / dt is not greater than 0.002%.

[0126] Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0127] Unless otherwise specified in the examples, the reaction temperature is room temperature, 20°C to 30°C.

[0128] The reaction progress in the embodiment is monitored by thin layer chromatography (TLC), and the developing solvent used in the reaction, the column chromatography eluent system used for purifying the compound and the developing solvent system of the thin layer chromatography include: A: dichloromethane / methanol system, the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid can also be added for adjustment.

[0129] Example 1

[0130] (S)-2-(1-(3-chlorophenyl)-2-hydroxyethyl)-6-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-1,2-dihydro-3H-pyrrolo[1,2-c]imidazol-3-one 1

[0131]

[0132] first step

[0133] (S)-2-((tert-Butyldimethylsilyl)oxy)-1-(3-chlorophenyl)ethylamine 1b

[0134] Dissolve (S)-2-amino-2-(3-chlorophenyl)ethanol 1a (4 g, 23.3 mmol, Shanghai Bid Pharmaceutical Technology Co., Ltd.) and imidazole (3.2 g, 46.6 mmol) in 80 mL of dichloromethane, add tert-butyldimethylsilyl chloride (5.2 g, 35 mmol) under ice bath, and stir to react for 14 hours. Add water, extract with dichloromethane (80 mL×3). Combine the organic phases, wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify by column chromatography with eluent system C to obtain the title compound 1b (6.5 g), yield: 97%.

[0135] MS m / z(ESI):286.1[M+1]

[0136] Step 2

[0137] (S)-N-((4-bromo-1H-pyrrol-2-yl)methyl)-2-((tert-butyldimethylsilyl)oxy)-1-(3-chlorophenyl)ethylamine 1d

[0138] 4-Bromo-1H-pyrrol-2-carboxaldehyde 1c (2.37 g, 13.62 mmol, Shanghai Bidex) and compound 1b (3.9 g, 13.64 mmol) were stirred and reacted for 3 hours. 100 mL of methanol was added for dilution, the temperature was lowered to 0°C, sodium borohydride (516 mg, 13.64 mmol) was added and stirred and reacted for 2 hours. Water was added, the reaction solution was concentrated under reduced pressure, water was added, and ethyl acetate was extracted (40 mL×3). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and purified by column chromatography with eluent system C to obtain the title compound 1d (4.8 g), with a yield of 79%.

[0139] MS m / z(ESI):444.2[M+1]

[0140] Step 3

[0141] (S)-6-Bromo-2-(2-((tert-butyldimethylsilyl)oxy)-1-(3-chlorophenyl)ethyl)-1H-pyrrolo[1,2-c]imidazol-3(2H)-one 1e

[0142] Compound 1d (4.8 g, 10.81 mmol) was dissolved in 100 mL of tetrahydrofuran, N, N'-carbonyldiimidazole (2.45 g, 15.11 mmol) was added under ice bath and stirred for 0.5 hours, sodium hydride (60%, 621 mg, 16.22 μmol) was added and stirred at room temperature for 14 hours. Saturated ammonium chloride was added. The reaction solution was concentrated under reduced pressure and purified by column chromatography with eluent system C to obtain the title compound 1e (4.0 g), with a yield of 78%.

[0143] MS m / z(ESI):469.1[M+1]

[0144] Step 4

[0145] (S)-2-(2-((tert-butyldimethylsilyl)oxy)-1-(3-chlorophenyl)ethyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[1,2-c]imidazol-3(2H)-one 1f

[0146] Under argon atmosphere, compound 1e (4.0 g, 8.51 mmol) was dissolved in 50 mL of 1,4-dioxane, and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-di(1,3,2-dioxaborolane) (3.24 g, 12.76 mmol), potassium acetate (3.34 g, 34.04 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]palladium trichloride (1.24 g, 1.70 mmol) were added in sequence, and stirred at 90°C for 2 hours. The mixture was cooled, filtered through diatomaceous earth, and the filtrate was concentrated and purified by column chromatography with eluent system C to give the title compound 1f (2.0 g) in a yield of 45%.

[0147] MS m / z(ESI):517.2[M+1]

[0148] Step 5

[0149] 4-Chloro-N-(1-methyl-1H-pyrazol-5-yl)pyrimidin-2-amine 1i

[0150] N-(1-methyl-1H-pyrazol-5-yl)formamide 1h (324.82 mg, 2.60 mmol, prepared by the method disclosed in patent application "WO2017 / 80979") was dissolved in 15 mL of N, N-dimethylformamide, sodium hydride (60%, 311.47 mg, 7.79 mmol) was added at 0°C, stirred for 0.5 hours, 4-chloro-2-(methylsulfonyl)pyrimidine 1g (500 mg, 2.60 mmol) was added, and the reaction was continued for 2 hours. 20 mL of water was added, and ethyl acetate was extracted (20 mL×3), the organic phases were combined and concentrated under reduced pressure, and the residue was purified by thin layer chromatography with developing solvent system C to obtain the title compound 1i (270 mg), with a yield of 49.6%.

[0151] MS m / z(ESI):210.3[M+1]

[0152] Step 6

[0153] (S)-2-(2-((tert-butyldimethylsilyl)oxy)-1-(3-chlorophenyl)ethyl)-6-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-1,2-dihydro-3H-pyrrolo[1,2-c]imidazol-3(2H)-one 1j

[0154] Under argon atmosphere, compound 1f (98.6 mg, 0.19 mmol), pre-prepared 4-chloro-N-(1-methyl-1H-pyrazol-5-yl)pyrimidin-2-amine 1i, [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (28 mg, 0.02 mmol), cesium carbonate (124 mg, 0.2 mmol) mixture was suspended in 20 mL 1,4-dioxane and 4 mL water, heated to 80°C and stirred for 14 hours. Cooled, filtered through diatomaceous earth, the filtrate was collected, extracted with ethyl acetate (20 mL×3), the organic phases were combined, concentrated under reduced pressure, and purified by column chromatography with eluent system A to obtain the title compound 1j (100 mg), yield: 92%.

[0155] MS m / z(ESI):564.3[M+1]

[0156] Step 7

[0157] (S)-2-(1-(3-chlorophenyl)-2-hydroxyethyl)-6-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-1,2-dihydro-3H-pyrrolo[1,2-c]imidazol-3-one 1

[0158] Compound 1j (100 mg, 0.17 mmol) was dissolved in 20 mL of dichloromethane, and 1 mL of trifluoroacetic acid was added dropwise. After the addition was completed, the mixture was stirred and reacted for 4 hours. The pH was adjusted to 7 with saturated sodium bicarbonate, and extracted with dichloromethane (20 mL × 2). The organic phases were combined, concentrated under reduced pressure, and purified by column chromatography with eluent system A to obtain the title compound 1 (15 mg). The yield was 18%. The product was detected by X-ray powder diffraction and was amorphous. The XRPD spectrum was as follows: Figure 1 .

[0159] MS m / z(ESI):450.1[M+1]

[0160] 1H NMR (400 MHz, CDCl 3): δ8.33(d, 1H), 7.72(s, 1H), 7.48(d, 1H), 7.41-7.33(m, 3H), 7.28-7.24(m, 1H), 7.18(s, 1H), 6.92(d, 1 H), 6.51 (s, 1H), 6.32 (d, 1H), 5.17 (dd, 1H), 4.46 (d, 1H), 4.32 (dd, 1H), 4.27-4.17 (m, 3H), 3.82 (s, 3H).

[0161] Biological evaluation

[0162] Test Example 1: ERK1 enzymatic activity test

[0163] 1. Test Purpose

[0164] The purpose of this experiment is to detect the inhibitory ability of the compounds on ERK1 enzyme activity. 50 The size of the compound was used to evaluate the in vitro activity. TM Kinase Assay Kit: Under the action of enzyme, the substrate is phosphorylated and ADP is produced. ADP-Glo ​​reagent is added to remove the unreacted ATP in the reaction system, and the Kinase detection reagent is used to detect the ADP produced by the reaction. In the presence of the compound, the inhibition rate of the compound is calculated by measuring the signal value.

[0165] 2. Experimental Methods

[0166] Enzyme and substrate preparation: ERK1 (1879-KS-010, R&D) and substrate (AS-61777, anaspec) were prepared in buffer to 0.75 ng / μL and 100 μM, respectively, and then the enzyme solution and substrate solution were prepared into a mixed solution at a volume ratio of 2:1 for standby use. ATP was diluted to 300 μM with buffer, and the compound was dissolved with DMSO (dimethyl sulfone, Shanghai Titan Technology Co., Ltd.) to prepare a storage solution with an initial concentration of 20 mM, and then the compound was prepared with Bravo. Finally, 3 μL of the mixed solution of enzyme and substrate, 1 μL of different concentrations of compounds (starting concentration of 50 μM, 4-fold dilution) were added to each well of the 384-well plate, and incubated at 30°C for 10 minutes. Finally, 1 μL of 300 μM ATP solution was added to each well and incubated at 30°C for 2 hours. Then, 5 μL of ADP-Glo ​​was added and incubated at 30°C for 40 minutes, followed by 10 μL of Kinase detection buffer and incubated at 30°C for 40 minutes. The 384-well plate was taken out and placed in an ELISA reader (BMG labtech, PHERAstar FS) to measure chemiluminescence using an ELISA reader.

[0167] 3. Data Analysis

[0168] The data were processed and analyzed using Microsoft Excel and Graphpad Prism 5. The IC values ​​of the compounds were obtained. 50 The results are shown in Table 1 below.

[0169] Table 1. IC values ​​of the compounds disclosed herein for inhibition of ERK1 enzyme activity 50 value

[0170] Example No. <![CDATA[IC 50 (nM)]]> 1 5

[0171] Conclusion: The disclosed compounds have a significant inhibitory effect on ERK1 enzyme activity.

[0172] Test Example 2: ERK2 Enzyme Activity Test

[0173] 1. Test Purpose

[0174] The purpose of this experiment is to detect the inhibitory ability of compounds on ERK2 enzyme activity. 50 The size of the compound was used to evaluate the in vitro activity. TM Kinase Assay Kit: Under the action of enzyme, the substrate is phosphorylated and ADP is produced. ADP-Glo ​​reagent is added to remove the unreacted ATP in the reaction system, and the Kinase detection reagent is used to detect the ADP produced by the reaction. In the presence of the compound, the inhibition rate of the compound is calculated by measuring the signal value.

[0175] 2. Experimental Methods

[0176] Enzyme and substrate preparation: ERK2 (1230-KS-010, R&D) and substrate (custom peptide, Gill Biochemical) were mixed in a buffer (40 mM Tris, 20 mM MgCl 2, 0.1mg / ml BSA, 50μM DTT) to 0.75ng / μL and 1500μM, then the enzyme solution and substrate solution were prepared into a mixed solution at a volume ratio of 2:1 for standby use. ATP was diluted to 500μM with buffer, the compound was dissolved with DMSO (dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.), and a storage solution with an initial concentration of 20mM was prepared, and then the compound was prepared with Bravo. Finally, 3μL of the mixed solution of enzyme and substrate, 1μL of compounds of different concentrations (starting concentration of 50μM, 4-fold dilution) were added to each well of the 384-well plate in sequence, incubated at 30℃ for 10 minutes, and finally 1μL of 500μM ATP solution was added to each well and incubated at 30℃ for 2 hours. Then 5μL of ADP-Glo ​​was added and incubated at 30℃ for 40 minutes, followed by 10μL of Kinase detection buffer and incubated at 30℃ for 40 minutes. The 384-well plate was taken out and placed in an ELISA reader (BMGlabtech, PHERAstar FS), and the chemiluminescence was measured using the ELISA reader.

[0177] 3. Data Analysis

[0178] The data were processed and analyzed using Microsoft Excel and Graphpad Prism 5. The IC values ​​of the compounds were obtained. 50 The results are shown in Table 2 below.

[0179] Table 2. IC values ​​of the compounds disclosed herein for inhibition of ERK2 enzyme activity 50 value

[0180] Example No. <![CDATA[IC 50 (nM)]]> 1 7

[0181] Conclusion: The disclosed compounds have a significant inhibitory effect on ERK2 enzyme activity.

[0182] Test Example 3: In vitro proliferation inhibition test of compounds on Colo205 tumor cells

[0183] 1. Test Purpose

[0184] The purpose of this experiment is to detect the inhibitory activity of compounds on the proliferation of Colo205 cells (CCL-222, ATCC) in vitro. The cells were treated with different concentrations of compounds in vitro and cultured for 3 days. Luminescent Cell Viability Assay (Promega, Catalog No.: G7573) was used to detect cell proliferation. 50 The values ​​were used to evaluate the in vitro activity of the compound.

[0185] 2. Experimental Methods

[0186] The following is an example of the in vitro proliferation inhibition test method for Colo205 cells to illustrate the method of testing the in vitro proliferation inhibition activity of the disclosed compounds on tumor cells in the present disclosure. This method is also applicable to, but not limited to, in vitro proliferation inhibition activity tests on other tumor cells.

[0187] Colo205 was digested, centrifuged and resuspended, the single cell suspension was mixed, and the live cell density was adjusted to 5.0×10 with cell culture medium (RPMI1640+2% FBS). 4 cells / ml, add 95 μl / well to a 96-well cell culture plate. Add only 100 μL of culture medium to the peripheral wells of the 96-well plate. Incubate the culture plate in an incubator for 24 hours (37°C, 5% CO 2 ).

[0188] Dissolve the compound in DMSO (dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.) and prepare a storage solution with an initial concentration of 20mM. The starting concentration of the small molecule compound is 2mM, 4-fold dilution, 9 dilution points, and the tenth point is DMSO. Take another 96-well plate, add 90μL of cell culture medium (RPMI1640+2% FBS) to each well, then add 10μL of different concentrations of the sample to be tested to each well, mix well, and then add 5μl of different concentrations of the sample to be tested to the cell culture plate, and duplicate wells for each sample. Incubate the culture plate in an incubator for 3 days (37°C, 5% CO 2 ). Take out the 96-well cell culture plate, add 50 μl of CTG solution to each well, and incubate at room temperature for 10 minutes. Measure chemiluminescence with an enzyme-labeled instrument (BMG labtech, PHERAstar FS).

[0189] 3. Data Analysis

[0190] The data were processed and analyzed using Microsoft Excel, Graphpad Prism 5. The results of the embodiment are shown in Table 3 below.

[0191] Table 3. IC values ​​of the compounds disclosed herein for inhibiting proliferation of Colo205 tumor cells in vitro 50 value

[0192] Example No. <![CDATA[IC 50 (nM)]]> 1 62

[0193] Pharmacokinetic evaluation

[0194] Test Example 4: Pharmacokinetics of the Disclosed Compounds in Mice

[0195] 1. Summary

[0196] Mice were used as test animals, and the drug concentrations in plasma at different times after oral administration of the compounds of Example 3, Example 10, Example 15 and Example 20 were determined by LC / MS / MS. The pharmacokinetic behavior of the disclosed compounds in mice was studied, and their pharmacokinetic characteristics were evaluated.

[0197] 2. Experimental plan

[0198] 2.1 Trial Drugs

[0199] Example 1 Compound.

[0200] 2.2 Experimental animals

[0201] Thirty-six female C57 mice were equally divided into four groups and purchased from Shanghai Jiesijie Experimental Animal Co., Ltd., with animal production license number: SCXK (Shanghai) 2013-0006.

[0202] 2.3 Drug preparation

[0203] A certain amount of compound was weighed, 5% volume of DMSO and 5% Tween 80 were added to dissolve it, and then 90% physiological saline was added to prepare a 0.1 mg / ml colorless clear solution.

[0204] 2.4 Administration

[0205] C57 mice were fasted overnight and then gavage was used to administer the drug. The dosage was 2 mg / kg and the administration volume was 0.2 ml / 10 g.

[0206] 3. Operation

[0207] Mice were given the drug by gavage, and 0.1 ml of blood was collected before and 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 11.0, and 24.0 hours after administration. The blood was placed in a heparinized tube and centrifuged at 3500 rpm for 10 minutes to separate the plasma, which was then stored at -20°C.

[0208] Determine the content of the test compound in mouse plasma after injection of different concentrations of the drug: take 25 μl of mouse plasma at each time after administration, add 50 μl (100 ng / mL) of internal standard solution camptothecin (China National Institute for the Control of Biological Products) and 200 μl of acetonitrile, vortex mix for 5 minutes, centrifuge for 10 minutes (4000 rpm), and take 4 μl of the supernatant of the plasma sample for LC / MS / MS analysis.

[0209] 4. Pharmacokinetic parameter results

[0210] Table 4. Pharmacokinetic parameters of the disclosed compounds are as follows:

[0211]

[0212]

[0213] Conclusion: The disclosed compounds have good pharmacokinetic absorption and pharmacokinetic advantages.

[0214] Example 2, Preparation of Fumarate I Crystalline Form

[0215] The amorphous compound of formula (I) (100 mg, 222.28 μmol) was added to 2 mL of ethyl acetate, heated to 60°C, 29 mg of fumaric acid was added, stirred for 30 minutes, gradually cooled to room temperature, and stirred at room temperature for 2 hours. Solids were gradually precipitated, filtered, and the filter cake was collected. The filter cake was washed with a small amount of ethyl acetate and vacuum dried at 40°C to obtain a solid (101.1 mg, yield: 80.4%). The obtained product 1 H-NMR data are shown below. The molar ratio of the compound represented by formula (I) to fumaric acid in the salt is 1:1.26

[0216] 1 H NMR (400 MHz, DMSO-d 6 )δ13.13(br,2H),9.32(s,1H)8.37(d,1H),7.92(s,1H),7.48(d,1H),7.41-7.33(m,3H),7.28-7.24(m,1H),7.18(s,1H),6. 66 (d, 1H), 6.63 (s, 1H), 6.27 (s, 1H), 5.26 (s, 1H), 5.17 (dd, 1H), 4.66 (d, 1H), 4.42 (dd, 1H), 3.94-4.06 (m, 2H), 3.82 (s, 3H).

[0217] The product was defined as Form I by X-ray powder diffraction detection, and the XRPD spectrum was as follows: Figure 2 , the peak positions are shown in Table 5.

[0218] Table 5. Fumarate I crystal peak position

[0219]

[0220] Example 3. Preparation of Fumarate II Crystalline Form

[0221] The amorphous compound of formula (I) (100 mg, 222.28 μ μmol) was added to 2 mL of ethanol, heated to 60°C, 29 mg of fumaric acid was added, stirred for 30 min, cooled to room temperature, and stirred for 2 hours. Solids were gradually precipitated, filtered, and the filter cake was collected. The filter cake was washed with a small amount of ethanol and dried in vacuo at 40°C. A solid (80 mg, yield: 63.6%) was obtained. The obtained product 1H-NMR data are shown below. The data show that the molar ratio of the compound represented by formula (I) in the salt to fumaric acid is 1:0.93.

[0222] 1 H NMR (400 MHz, DMSO-d 6 ): δ13.13(br,2H), 9.32(s, 1H), 8.37(d, 1H), 7.92(s, 1H), 7.48(d, 1H), 7.41-7.33(m, 3H), 7.28-7.24(m, 1H), 7.18(s, 1H), 6. 66 (d, 1H), 6.63 (s, 1H), 6.27 (s, 1H), 5.26 (s, 1H), 5.17 (dd, 1H), 4.66 (d, 1H), 4.42 (dd, 1H), 3.94-4.06 (m, 2H), 3.82 (s, 3H).

[0223] The product was defined as Form II by X-ray powder diffraction detection, and the XRPD spectrum was as follows: Figure 3 The peak positions are shown in Table 6.

[0224] Table 6. Fumarate II crystal peak position

[0225]

[0226] DSC spectrum Figure 4 The endothermic peaks are 58.80℃ and 161.41℃; the TGA spectrum is as follows Figure 5 , 25℃-130℃, weight loss is 1.52%, 130℃-280℃, weight loss is 14.54%.

[0227] DVS test shows that under normal storage conditions (i.e. 25°C, 60% RH), the sample has a moisture absorption weight gain of about 2.38%; under accelerated test conditions (i.e. 70% RH), the moisture absorption weight gain is about 2.75%; under extreme conditions (90% RH), the moisture absorption weight gain is about 3.85%. During the humidity change from 0% to 95% RH, the desorption process of the sample does not overlap with the adsorption process (see Figure 6 After DVS testing, the crystal form was retested and the crystal form did not change (see Figure 7 ).

[0228] Example 4. Preparation of Fumarate III Crystalline Form

[0229] The amorphous compound of formula (I) (100 mg, 222.28 μmol) was added to 5 ml of acetonitrile, and then 27.0 mg of fumaric acid was added, stirred at 50° C. for 12-36 h, filtered, and dried in vacuo at 40° C. The product was defined as Form III by X-ray powder diffraction detection. The XRPD spectrum is shown in Figure 8, the peak positions are shown in Table 7. The NMR data show that the molar ratio of the compound represented by formula (I) to fumaric acid in the salt is 2:1.

[0230] Table 7. Fumarate III crystal peak position

[0231]

[0232] Example 5. Preparation of Fumarate IV Crystalline Form

[0233] 100 mg of the fumarate III crystal sample was placed in 93% RH overnight, and the product was defined as crystal IV by X-ray powder diffraction detection. The XRPD spectrum is as follows: Fig. 9 The peak positions are shown in Table 8. The TGA spectrum shows that the crystal loses 0.73% weight between 30-100°C and 10.89% weight between 120-200°C; the DSC spectrum shows that the crystal has an endothermic peak with a peak value of 148.3°C. The nuclear magnetic resonance data show that the molar ratio of the compound represented by formula (I) to fumaric acid in the salt is 2:1.

[0234] Table 8. Fumarate Form IV Peak Position

[0235]

[0236]

[0237] Example 6. Preparation of Form a of Oxalate

[0238] Weigh 100 mg of the amorphous compound represented by formula (I), add 10.5 mg of oxalic acid and 5 ml of hexane, stir at 50°C for 24-36 hours, filter, and vacuum dry at 40°C to obtain a solid. The product is defined as crystal form a by X-ray powder diffraction detection. The XRPD spectrum is as follows: Fig.10 , the peak positions are shown in Table 9.

[0239] Table 9. Peak position of a-form of oxalate

[0240]

[0241] Example 7. Preparation of maleate salt crystalline form a

[0242] The amorphous compound of formula (I) (100 mg, 222.28 μmol) was added to 2 mL of isopropanol, heated to 60°C, and 29 mg of maleic acid was added. The mixture was stirred at 60°C for 30 minutes, cooled to room temperature, and stirred at room temperature for 2 hours. A small amount of solid was gradually precipitated. The mixture was stirred overnight at room temperature. More solid was precipitated. The filter cake was collected by suction filtration and washed with a small amount of isopropanol. The product was dried under vacuum at 40°C to obtain 90.1 mg of the product. 1H-NMR data are shown below. The molar ratio of the compound represented by formula (I) to maleic acid in the salt is 1:0.95.

[0243] 1 H NMR (400 MHz, DMSO-d 6 ): 9.33 (s, 1H) 8.37 (d, 1H), 7.92 (s, 1H), 7.48 (d, 1H), 7.41-7.33 (m, 3H), 7.28-7.24 (m, 1H), 7.18 (s, 1H) ), 6.66(s, 1H), 6.27(s, 2H), 5.17(dd, 1H), 4.66(d, 1H), 4.42(dd, 1H), 3.94-4.06(m, 2H), 3.69(s, 3H).

[0244] The product was defined as Form a by X-ray powder diffraction detection, and the XRPD spectrum was as follows: Fig.11 , the peak positions are shown in Table 10.

[0245] Table 10. Peak positions of maleate a crystal form

[0246]

[0247]

[0248] Example 8. Preparation of maleate b crystal form

[0249] The amorphous compound of formula (I) (100 mg, 222.28 μmol) was added to 2 mL of ethyl acetate, heated to 60°C, and 29 mg of maleic acid was added. The mixture was stirred at 60°C for 30 minutes, cooled to room temperature, and stirred at room temperature for 2 hours. Solids gradually precipitated, and the mixture was stirred at room temperature overnight. The mixture was filtered, the filter cake was collected, and washed with a small amount of ethyl acetate. The product was dried in vacuo at 40°C to obtain 125.1 mg of the product.

[0250] The product 1 H-NMR data are shown below. The data show that the molar ratio of the compound represented by formula (I) to maleic acid in the salt is 1:0.97.

[0251] 1H NMR (400 MHz, DMSO-d 6): 9.33(s, 1H), 8.37(d, 1H), 7.92(s, 1H), 7.48(d, 1H), 7.41-7.33(m, 3H), 7.28-7.24(m, 1H), 7.18(s, 1 H), 6.66 (s, 1H), 6.27 (s, 2H), 5.17 (dd, 1H), 4.66 (d, 1H), 4.42 (dd, 1H), 3.94-4.06 (m, 2H), 3.69 (s, 3H).

[0252] The product was defined as Form b by X-ray powder diffraction detection, and the XRPD spectrum was as follows: Fig.12 , the peak positions are shown in Table 11.

[0253] Table 11. Peak positions of maleate b crystal form

[0254]

[0255] Example 9. Preparation of maleate c crystal form

[0256] 100 mg of the amorphous compound represented by formula (I) was added with 13.5 mg of maleic acid and 2 ml of acetonitrile, stirred at 50°C for 3 h, filtered, and dried in vacuo at 40°C to obtain a solid. The obtained product 1 H-NMR is shown below. The nuclear magnetic data show that the molar ratio of the compound represented by formula (I) to maleic acid in the salt is: 1:1

[0257] 1 H NMR (400 MHz, DMSO-d 6 ): 9.33(s, 1H), 8.37(d, 1H), 7.92(s, 1H), 7.48(d, 1H), 7.41-7.33(m, 3H), 7.28-7.24(m, 1H), 7.18(s, 1 H), 6.66(s, 1H), 6.27(s, 2H), 5.17(dd, 1H), 4.66(d, 1H), 4.42(dd, 1H), 3.94-4.06(m, 2H), 3.69(s, 3H)

[0258] The product was defined as Form C by X-ray powder diffraction detection. The XRPD spectrum is as follows: Fig.13 , the peak positions are shown in Table 12.

[0259] Table 12. Peak positions of maleate c crystalline form

[0260]

[0261] Example 10: Preparation of amorphous maleate

[0262] The free base of the compound represented by formula (I) (500 mg, 1.11 mmol) was added to 10 mL of methanol, and 129 mg of maleic acid was added. The temperature was raised to 60° C., stirred for 30 min, and rotary evaporated to obtain the title product (605 mg, yield: 96.2%).

[0263] The product 1 H-NMR data are shown below. The H-NMR data indicate that the molar ratio of the compound represented by formula (I) to maleic acid in the salt is 1:1.

[0264] 1 H NMR (400 MHz, DMSO-d 6 ): 9.33 (s, 1H) 8.37 (d, 1H), 7.92 (s, 1H), 7.48 (d, 1H), 7.41-7.33 (m, 3H), 7.28-7.24 (m, 1H), 7.18 (s, 1 H), 6.66(s, 1H), 6.27(s, 2H), 5.17(dd, 1H), 4.66(d, 1H), 4.42(dd, 1H), 3.94-4.06(m, 2H), 3.69(s, 3H)

[0265] The product was amorphous as determined by X-ray powder diffraction. Fig.14 .

[0266] Example 11. Preparation of maleate d crystal form

[0267] The amorphous maleate salt of the compound represented by formula (I) (Example 10) (36 mg, 63.6 μmol) was added to 0.4 mL of toluene, stirred at room temperature for 72 hours, filtered, the filter cake was collected, and vacuum dried at 40°C to obtain the title product (26.0 mg, yield: 73.4%).

[0268] The product 1 H-NMR data are shown below. The H-NMR data indicate that the molar ratio of the compound represented by formula (I) to maleic acid in the salt is 1:1.

[0269] 1H NMR (400 MHz, DMSO-d 6 ): 9.31 (s, 1H) 8.35 (d, 1H), 7.91 (s, 1H), 7.48 (d, 1H), 7.41-7.33 (m, 3H), 7.28-7.24 (m, 1H), 7.15 (s, 1H) ), 6.63(s, 1H), 6.27(s, 2H), 5.17(dd, 1H), 4.63(d, 1H), 4.42(dd, 1H), 3.94-4.06(m, 2H), 3.71(s, 3H).

[0270] The product was defined as Form d by X-ray powder diffraction detection. The XRPD spectrum is as follows: Fig.15 , the peak positions are shown in Table 13.

[0271] Table 13. Peak positions of maleate d crystal form

[0272]

[0273] Example 12: Preparation of Maleate Crystal Form e

[0274] The maleate c crystal form was placed in an oven at 60°C overnight to obtain a solid. The product was defined as crystal form e by X-ray powder diffraction detection. The XRPD spectrum is shown in Fig.16 , the peak positions are shown in Table 14.

[0275] Table 14. Peak positions of maleate e crystal form

[0276]

[0277] Example 13. Preparation of α-crystalline form of succinate

[0278] Weigh 100 mg of the free base of the compound represented by formula (I), add 13.8 mg of succinic acid and 5 ml of isopropyl ether, stir at 50°C for 12-36 h, filter, and dry in vacuo at 40°C. The product is defined as Form α. The XRPD spectrum is as follows: Fig.17 , the peak positions are shown in Table 15.

[0279] Table 15. Peak positions of α-form succinate

[0280]

[0281]

[0282] Example 14. Preparation of β-crystalline form of succinate

[0283] Weigh 100 mg of the free base of the compound represented by formula (I), add 13.8 mg of succinic acid and 5 ml of acetonitrile, stir at 50°C for 12-36 h, filter, and vacuum dry at 40°C. The product is defined as crystalline form β. The XRPD spectrum is as follows: Fig.18 , the peak positions are shown in Table 16.

[0284] Table 16. Peak positions of β-succinate crystals

[0285]

[0286] Example 15. Preparation of α-crystalline form of hydrobromide

[0287] Weigh 100 mg of the free base of the compound represented by formula (I), add 7 μL of hydrobromic acid and 5 ml of acetonitrile, stir at 50°C for 2-36 hours, filter, and vacuum dry at 40°C. The product is tested by ion chromatography, and the bromide ion content is 7.6%. The molar ratio of the compound represented by formula (I) to hydrobromic acid in the salt is 1:1. The product is defined as crystal form α. The XRPD spectrum is as follows: Fig.19 The peak positions are shown in Table 17. The TGA spectrum shows that the crystal loses 0.27% weight between 30-170°C and 14.47% weight between 190-290°C; the DSC spectrum shows that the crystal has an endothermic peak with a peak value of 175.85°C.

[0288] Table 17. Peak positions of hydrobromide α-form

[0289]

[0290]

[0291] Example 16. Preparation of β-crystalline form of hydrobromide

[0292] Weigh 100 mg of the free base of the compound represented by formula (I), add 14 μl of hydrobromic acid and 5 ml of acetonitrile, stir at 50°C for 2 h, filter, and vacuum dry at 40°C. The product is tested by ion chromatography, and the bromide ion content is 14.3%. The molar ratio of the compound represented by formula (I) to hydrobromic acid in the salt is 1:2. The product is defined as crystalline form β. The XRPD spectrum is as follows: Fig. 20 The peak positions are shown in Table 18. The TGA spectrum shows that the crystal loses 0.58% of its weight between 30-130°C and 17.26% of its weight between 150-290°C; the DSC spectrum shows that the crystal has an endothermic peak with a peak value of 213.86°C.

[0293] Table 18. Hydrobromide β-form peak position

[0294]

[0295] Example 17. Preparation of γ-crystalline form of hydrobromide

[0296] Weigh 100 mg of the free base of the compound represented by formula (I), add 14 μl of hydrobromic acid and 5 ml of acetonitrile, stir at 50°C for 36 h, filter, and vacuum dry at 40°C for 1 h. The product is defined as crystal form γ. The XRPD spectrum is as follows: Fig.21 , the peak positions are shown in Table 19.

[0297] Table 19. γ-crystal peak position of hydrobromide salt

[0298]

[0299] Example 18. Preparation of Form I of Hydrochloride

[0300] Weigh 100 mg of the free base of the compound represented by formula (I), add 20 μL of hydrochloric acid and 5 mL of acetonitrile, stir at 50°C for 36 h, filter, and vacuum dry at 40°C. The product is tested by ion chromatography, and the chloride ion content is 6.52%, indicating that the molar ratio of the compound represented by formula (I) to hydrochloric acid in the salt is 1:1, which is defined as crystal form I. The XRPD spectrum is as follows: Fig. 22 , the peak positions are shown in Table 20.

[0301] Table 20. Hydrochloride I crystal peak position

[0302]

[0303] Example 19. Preparation of Form II of Hydrochloride

[0304] Weigh 100 mg of the free base of the compound represented by formula (I), add 20 μL of hydrochloric acid and 5 mL of ethanol, stir at 50°C for 1 hour to clarify, cool at room temperature for 1 hour, stir at 25°C for 36 hours, filter, and vacuum dry at 40°C. The product is tested by ion chromatography, and the chloride ion content is 6.88%, indicating that the molar ratio of the compound represented by formula (I) to hydrochloric acid in the salt is 1:1, which is defined as crystal form II. The XRPD spectrum is as follows: Fig.23 , the peak positions are shown in Table 21.

[0305] Table 21. Hydrochloride II crystal peak position

[0306]

[0307] Example 20. Preparation of Form III of Hydrochloride

[0308] Weigh 100 mg of the free base of the compound represented by formula (I), add 10 μL of hydrochloric acid and 5 ml of ethanol, stir at 50°C for 1 hour to clarify, cool at room temperature for 1 hour, stir at 25°C for 36 hours, filter, and vacuum dry at 40°C. The product is tested by ion chromatography, and the chloride ion content is 6.82%, indicating that the molar ratio of the compound represented by formula (I) to hydrochloric acid in the salt is 1:1, which is defined as crystal form III. The XRPD spectrum is as follows: Fig.24 , the peak positions are shown in Table 22.

[0309] Table 22. Peak positions of hydrochloride III crystal form

[0310]

[0311]

[0312] Example 21. Preparation of Form IV of Hydrochloride

[0313] The hydrochloride salt form I was heated to 135°C (10K / min), and the product was defined as form IV. The XRPD spectrum is as follows Fig.25 , the peak positions are shown in Table 23.

[0314] Table 23. Hydrochloride IV Crystalline Peak Position

[0315]

[0316] Example 22. Preparation of Form V of Hydrochloride

[0317] Weigh 100 mg of the free base of the compound represented by formula (I), add 40 μL of hydrochloric acid and 10 mL of ethanol, stir at 50°C for 1 hour until clear, place at 25°C and stir for 3-12 hours, filter, and vacuum dry at 40°C. The product is defined as Form V. The XRPD spectrum is as follows: Fig.26 , the peak positions are shown in Table 24.

[0318] Table 24. Hydrochloride V Crystalline Peak Position

[0319]

[0320]

[0321] Example 23: Study on stability of influencing factors

[0322] The four crystal forms of hydrobromide α, hydrobromide β, fumaric acid II and fumaric acid IV were laid out in the open, and the stability of the samples was investigated under light (4500 Lux), high temperature (40°C, 60°C), and high humidity (RH 75%, RH 92.5%) conditions, and the sampling period was 30 days.

[0323] Table 25. Stability data of factors affecting the α-crystalline form of hydrobromide

[0324]

[0325]

[0326] Table 26. Stability data of factors affecting the β-form of hydrobromide

[0327]

[0328] Table 27. Results of 30-day test on factors affecting the crystal form of fumarate II

[0329]

[0330]

[0331] Table 28. Stability data of factors affecting the crystal form of fumarate IV

[0332]

[0333] Conclusion: The experiment of influencing factors showed that under high temperature of 40℃ and 60℃ and high humidity of 75% and 92.5%, the α-crystalline hydrobromide, β-crystalline hydrobromide, II-crystalline fumarate and IV-crystalline fumarate had good physical and chemical stability.

[0334] Example 24. Long-term / accelerated stability study

[0335] 1. The hydrobromide α crystal was placed at 25°C, 60% RH and 40°C, 75% RH to investigate its stability.

[0336] Table 29

[0337]

[0338] Long-term / accelerated stability experiments show that the hydrobromide α-crystal has good physical and chemical stability when placed for one month under long-term accelerated stability conditions.

[0339] 2. The hydrobromide β-crystal was placed at 25°C, 60% RH and 40°C, 75% RH to investigate its stability.

[0340] Table 30

[0341]

[0342] Long-term / accelerated stability experiments show that the hydrobromide β-crystal has good physical and chemical stability when placed for 1 month under long-term accelerated stability conditions.

[0343] 3. The fumarate IV crystal was placed at 25°C, 60% RH and 40°C, 75% RH to investigate its stability.

[0344] Table 31

[0345]

[0346]

[0347] Long-term / accelerated stability experiments show that the physical and chemical properties of the fumarate IV crystal form are stable after being placed under long-term accelerated stability conditions for one month.

[0348] 4. Fumarate Form II was subjected to long-term (25°C, 60% RH) and accelerated (40°C, 75% RH) stability studies for 6 months. The results are shown in the following table.

[0349] Table 32, long-term accelerated stability test results of fumarate crystal form II:

[0350]

[0351] Experimental conclusion: The results show that the fumarate crystal form II sample of the compound of formula 1 has good physicochemical stability when placed under long-term (25°C, 60% RH) and accelerated (40°C, 75% RH) conditions for 6 months.

[0352] Example 25. Preparation of amorphous fumarate

[0353] 500 mg of the free base of the compound represented by formula (I) was dissolved in 10 mL of methanol, 1 eq of fumaric acid and 60 eq of acid were added, and the mixture was stirred for 30 min, and then dried to obtain a solid, which was amorphous as determined by XRPD.

Claims

1. A pharmaceutically acceptable salt of a compound of formula (I), wherein the pharmaceutically acceptable salt is selected from fumarate, oxalate, succinate, maleate, hydrochloride or hydrobromide, 2. A fumarate salt of the compound of formula (I) according to claim 1, wherein the molar ratio of the compound of formula (I) to fumaric acid is 1:1 or 2:

1.

3. A crystal form I of a fumarate salt of a compound represented by formula (I), wherein The fumarate of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to fumaric acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 10.671, 22.735, 28.681, and 29.

302.

4. A crystal form II of a fumarate salt of a compound represented by formula (I), wherein The fumarate of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to fumaric acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 7.783, 9.151, 12.378, 14.808, 18.574, 25.755, and 26.

802.

5. A III crystalline form of a fumarate salt of a compound represented by formula (I), wherein The fumarate of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to fumaric acid is 2:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 4.877, 10.601, 13.827, 14.867, 16.272, 18.874, 23.921, and 26.

367.

6. The IV crystalline form of the fumarate of the compound represented by formula (I) according to claim 1, wherein the fumarate of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to fumaric acid is 2:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 4.854, 10.613, 13.692, 23.523, 24.252, and 25.

925.

7. A hydrobromide of the compound of formula (I) according to claim 1, wherein the molar ratio of the compound of formula (I) to hydrobromic acid is 1:1 or 1:

2.

8. An alpha crystalline form of the hydrobromide salt of the compound represented by formula (I), The hydrobromide of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to hydrobromic acid is 1:1, and the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 6.656, 9.552, 14.110, 19.095, 20.092, 25.220, and 27.

119.

9. A β-crystalline form of the hydrobromide salt of a compound represented by formula (I), The hydrobromide of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to hydrobromic acid is 1:2, has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, and has characteristic peaks at 7.363, 12.914, 15.392, 16.865, 17.217, 19.704, 21.999, 22.515, 24.769, 27.056, and 28.

236.

10. The crystalline form of the fumarate salt or the crystalline form of the hydrobromide salt of the compound of formula (I) according to any one of claims 3 to 6 and 8 to 9, wherein: The error range of the 2θ angle is ±0.

2.

11. A pharmaceutical composition comprising the following components: i) a pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 1 to 10, or a crystalline form thereof, or a mixture thereof; and ii) one or more pharmaceutically acceptable excipients.

12. A method for preparing the pharmaceutical composition according to claim 11, comprising the steps of mixing a pharmaceutically acceptable salt of a compound of formula (I) according to any one of claims 1 to 10, or a crystalline form thereof, or a mixture thereof with one or more pharmaceutically acceptable excipients, 13. Use of a pharmaceutically acceptable salt of a compound of formula (I) according to any one of claims 1 to 10, or a crystal form thereof, or a mixture thereof, or a pharmaceutical composition according to claim 11 in the preparation of a medicament for treating or preventing cancer, wherein the cancer is selected from colon cancer or rectal cancer.

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